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Environmental Microbiology August 2009, Volume 11, Issue 8, Pages 1983 - 1997
Archimer, archive institutionnelle de l’Ifremer http://www.ifremer.fr/docelec/
http://dx.doi.org/10.1111/j.1462-2920.2009.01921.x © 2009 Wiley Blackwell Publishing, Inc. The definitive version is available at http://www3.interscience.wiley.com/
Isolation and physiological characterization of two novel, piezophilic, thermophilic chemolithoautotrophs from a deep-sea hydrothermal vent chimney Ken Takai1, * Masayuki Miyazaki1, Hisako Hirayama1, Satoshi Nakagawa1, Joël Querellou2 and Anne Godfroy2 1
Sunground Animalcule Retrieval (SUGAR) Program, Japan Agency for Marine-Earth Science & Technology, 215 Natsushima-cho, Yokosuka 237-0061, Japan 2 Ifremer, Ctr Brest, Laboratoire de Microbiologie des Environnements Extrêmes, UMR 6197, F-29280 Plouzané, France. *: Corresponding author : Takai Ken, Tel. (+81) 468 67 9677; Fax (+81) 468 67 9715, email address :
[email protected]
Abstract: Two novel, thermophilic piezophiles, capable of chemolithoautotrophic growth, are successfully cultivated and isolated from a black smoker chimney at the TAG field (Mid Atlantic Ridge: MAR) by using a piezophilic cultivation technique. Both strains (strains 106 and 108) represent dominant cultivated populations of the microbial communities in the chimney surface habitat. Strain 106 represents typically thin, long spiral cells under the piezophilic growth condition but short bent cells under the non-piezophilic condition. It is a strictly chemolithoautotrophic gammaproteobacterium using reduced sulfur compounds as the electron donors, and nitrate and O2 as the electron acceptors. Based on the 16S rRNA gene sequence, strain 106 would represent a novel genus of the previously uncultivated group (Symbiont Group I; a potentially novel family) within the Gammaproteobacteria, and 'Thioprofundum lithotrophica' gen. nov., sp. nov. is proposed. Strain 108 is a short, oval rod at any of the growth pressures. It is a facultative chemoautotroph, capable of both chemolithoautotrophic growth with H2 and S oxidations and organotrophic growth with complex organics or organic acids using nitrate and O2 as the electron acceptors. The chemolithoautotrophic growth is strictly piezophilic and under the organotrophic growth condition, it grows at conventional pressures (0.1 MPa). Strain 108 is phylogenetically distinctive from any of the previously described genera of the family Rhodobacteraceae within the Alphaproteobacteria, and 'Piezobacter thermophilus' gen. nov., sp. nov. is proposed. The piezophilic cultivation technique can be a powerful tool to isolate and characterize the previously uncultivated phylotypes in the deep-sea hydrothermal vent environments.
1
43
INTRODUCTION
44 45
Research on deep-sea piezophiles has half century of history since the pioneering
46
work by Zobell & Morita (1957). For 50 years, most of the targeted microorganisms
47
have been heterotrophs, while very few piezophilic chemolithoautotrophs were
48
described (Bernhardt et al., 1988; Kato, 2006; Miller et al., 1988; Park et al., 2006).
49
Thermophilic hydrogenotrophic methanogens Methanocaldococcus jannaschii and
50
Methanothermococcus thermolithotrophicus were isolated from deep-sea and deep
51
subsurface environments originally by conventional, non-piezophilic cultivation
52
techniques but were later found to be facultative piezophilic based on the hyperbaric
53
pressure cultivation experiments (Bernhardt et al., 1988; Miller et al., 1988;). Recently,
54
a new hydrostatic pressure cultivation technique has been developed, which has enabled
55
cultivation of various types of deep-sea chemolithoautotrophs such as hydrogenotrophic
56
methanogen, sulfate-reducer and H2- and S-oxidizing nitrate- and O2-reducers
57
previously isolated by non-piezophilic techniques (Takai et al., 2008a). This technique
58
was successfully applicable even for the most hyperthermophilic microorganism,
59
Methanopyrus kandleri, which had been also known to be quite hard to cultivate (Takai
60
et al., 2008a). Nevertheless, all the deep-sea piezophilic chemolithoautotrophs studied
61
so far were initially isolated by the cultures under the conventional gas pressures (up to
62
0.4 MPa), and none of the chemolithoautotrophs is obtained by a high-pressure culture
63
directly from a deep-sea habitat.
64
Direct piezophilic enrichment from deep-sea and deep subsurface microbial
65
communities would be advantageous to obtain the numerically predominant or the
66
functionally significant microbial populations in the in situ habitats. This is not only 3
67
because the in situ hydrostatic pressure is a potential key physical parameter directly
68
affecting their growth (Abe et al., 1999; Kato, 2006) but also because the pressure and
69
temperature serve as the primary physicochemical factors preparing the thermodynamic
70
states of energy and carbon metabolisms for their growth. That is to say, using
71
conventional gas pressures of media, available gaseous substrates dissolved in the liquid
72
phase of microbial medium often become much less abundant as compared to the
73
concentrations in the in situ environments. For instances, the highest dissolved
74
concentrations of H2, N2, CH4 and CO2 in the deep-sea hydrothermal fluids are recorded
75
to be up to 16, 36, 54 and 2700 mM, respectively (Charlou et al., 2002; Lupton et al.,
76
2008; Person et al., 2005) but in the test tubes or bottles at 60 ˚C under a 0.2 MPa of gas
77
pressure, theoretically the soluble concentrations are less than 1.6, 1.2, 2.0 and 23.5 mM,
78
respectively. The different abundance of the gaseous energy and carbon sources may be
79
a great metabolic bias for enrichment of certain populations that are less predominant in
80
the in situ habitats but are energetically favorable in laboratory experiments. In contrast,
81
if the piezophilic cultivation provides quite dissimilar thermodynamic states compared
82
to those of the in situ habitats, it may lead to enrichment of previously uncultivated and
83
unidentified microbial components of the similar environments.
84
In
this
study,
we
tried
to
cultivate
H2-
and/or
sulfur-oxidizing
85
chemolithoautotrophs using the piezophilic cultivation technique from a black smoker
86
chimney obtained from the TAG field in the Mid Atlantic Ridge (MAR). The TAG field
87
is located at water depth of between 3620 and 3660 m and is among the world deepest
88
hot hydrothermal systems. The H2- and/or sulfur-oxidizing chemolithoautotrophs are
89
one of the most predominant microbial populations in the global deep-sea hydrothermal
90
environments and a diversity of Aquificae and Epsilonproteobacteria members have 4
91
been isolated as the primary components (Takai et al., 2006a; Nakagawa and Takai
92
2006; 2008). Although only one pressure-temperature (PT) condition of 50 ˚C and 36
93
MPa and only one medium was used in this study, two novel chemolithoautotrophic
94
bacteria were enriched and isolated by purification under different pressure conditions.
95
These piezophilic chemolithoautotrophs represented the previously uncultivated
96
phylotypes of Proteobacteria. The physiological properties of the new deep-sea
97
piezophiles were characterized under the piezophilic cultivation condition.
98
5
98
MATERIALS AND METHODS
99 100
Sample collection and fluid chemistry: A sample from a black smoker chimney
101
was obtained from the TAG hydrothermal field (26˚08.23N, 44˚49.57W) in the Mid
102
Atlantic Ridge (MAR) at a depth of 3626 m by means of the remotely operative vehicle
103
VICTOR6000 (Ifremer) in Dive#263 during the EXOMAR cruise performed in August
104
2005. This chimney was one of the numerous chimneys comprising so-called “the black
105
smoker complex (BSC)” in the TAG field (e.g., Charlou et al., 1996). The temperature
106
of the black smoker fluid hosted by this chimney (named as Matomo chimney) was
107
measured to be maximally 345 ˚C. The chimney portions were broken by a manipulator
108
of the VICTOR6000 and directly dropped into a sample box that had been in advance
109
decontaminated and filled with the sterilized distilled water (Postec et al., 2005). The lid
110
of the sample box was closed immediately after the sampling at the seafloor although
111
the box did not preserve the hydrostatic pressure. However, due to the tightly sealed lid,
112
no additional seawater mixing was expected during returning to sea surface.
113
Immediately after the recovery of the chimney sample onboard, a relatively large
114
piece of structure, which preserved the intact structure as much as possible, was
115
subsampled into the representative microbial habitats as previously described (Takai et
116
al., 2001; 2008b). The chimney outer surface (thickness 1 to 2 mm) and chimney inside
117
part (thickness 1 to 2 cm) were collected for the nucleic acid extraction, the microscopic
118
observation and the cultivation. For the subsamples of the cultivation, each of the outer
119
surface (2 g wet weight) and the inside part (40 g wet weight) was suspended in 20 ml
120
of seawater filtered with a 0.22 µm pore size filter in the presence and absence of 0.05%
121
(w/v) neutralized sodium sulfide in a 100 ml glass bottle (Schott Glaswerke, Mainz, 6
122
Germany), and then tightly sealed with a butyl rubber cap under a gas phase of 100% N2
123
(0.2 MPa). For the subsamples of the microscopic observation, approx. 1 g (wet weight)
124
of the subsample was fixed with 3 ml of filter-sterilized seawater containing 3.7% (v/v)
125
formalin for 24 h and then stored at -80 ˚C. All the samples for cultivation were
126
transferred to our laboratory under refrigeration and were preserved at 4 ˚C prior to
127
experiments.
128
The hydrothermal fluid chemistry in the TAG field is already characterized in
129
detail (Campbell et al., 1988; Charlou et al., 1996; Chiba et al., 2001). It is known that
130
the hydrothermal fluids emitting from the BSC of the TAG field have one endmember
131
fluid source (Chiba et al., 2001). The chemical composition of the endmember fluid is
132
determined and is found to be temporally stable (Campbell et al., 1988; Charlou et al.,
133
1996; Chiba et al., 2001).
134
Total direct cell counts: Microbial community densities in the chimney
135
subsamples were determined by 4',6-diamidino-2-phenylindole (DAPI)-staining direct
136
count. The frozen formalin-fixed chimney subsample was thawed, and then vigorously
137
suspended with a vortex mixer. After 5 min of static state, 0.5 ml of formalin-fixed
138
supernatant was added to 0.5 ml of filter-sterilized phosphate-buffered saline (PBS, pH
139
7.2) containing DAPI (10 µg/ml), and incubated at room temperature for 30 min. After
140
the mixture was filtered, each filter was rinsed twice with 2 ml of filter-sterilized PBS.
141
The filters were examined under epifluorescence using a phase-contrast Olympus BX51
142
microscope with the Olympus DP71 digital camera system. An average total cell count
143
was obtained from more than 100 microscopic fields from three separate filters.
144
Liquid serial dilution cultures: To estimate the abundance of culturable
145
microorganisms (viable counts) represented by a variety of physiological and metabolic 7
146
characteristics, a series of serial dilution cultures were done from the chimney
147
subsamples under the various cultivation conditions, which were in a same manner as
148
previously described (e.g., Takai et al., 2008b). Since the detail methods and results will
149
be described elsewhere, only the media and culture conditions that gave positive
150
cultivation
151
sulfur-reducing thermophiles such as members of Thermococcales and Thermotogales,
152
MJYPS medium (Takai et al., 2000) was used (Table 1). For hydrogen- and/or
153
sulfur-oxidizing
154
Gammaproteobacteria and Epsilonproteobacteria, MMJHS medium (Takai et al. 2003)
155
was used (Table 1). For anaerobic dissimilatory Fe(III)- and/or sulfate-reducers, such as
156
members
157
Deltaproteobacteria, a MMJHFe medium (Takai et al. 2008b) was used (Table 1).
results
of
were
described
(Table
chemolithoautotrophs,
Archaeoglobales,
such
1).
as
Deferribacteres,
For
anaerobic,
members
of
heterotrophic
Aquificales,
Thermodesulfobacteria
and
158
A serial dilution culture using a piezophilic cultivation technique (Takai et al.,
159
2008a) was also performed with the chimney surface sample at 50 ˚C and at 36 MPa
160
(Table 1). The detail procedure is described below.
161
The microbial growth was identified by turbidity and/or microscopic observation
162
for maximally 2 months. The microorganism present in the most diluted series of the
163
medium at each temperature was isolated by the subsequent extinction-dilution method
164
(Takai and Horikoshi, 2000). The partial sequences of the 16S rRNA genes (approx.
165
700-1000 bp) of the isolates were determined as described elsewhere (Takai et al..
166
2004).
167
Enrichment and purification with piezophilic cultivation techniques: The
168
chimney surface slurry preserved in the absence of sodium sulfide was inoculated into
169
the test tubes containing MMJHS medium (Takai et al., 2003) under a gas phase of 80% 8
170
H2 + 20% CO2 (0.2 MPa) with every 1/5 dilution. A 1 ml portion of the inoculated
171
culture was taken from each of the serial dilution of test tubes into a piezophilic
172
cultivation syringe and then, a 250 µl of 100% H2 was added to the syringe
173
(corresponding to approx. 10 mM of soluble H2 and 30 mM of ΣCO2 under the
174
piezophilic condition) (Takai et al., 2008a). The syringe was sealed by a needle sticking
175
into a butyl rubber stopper (Takai et al., 2008a). Finally, the pressure vessels containing
176
the cultivation syringes were compressed by a hydraulic pomp at 36 MPa and incubated
177
at 50 ˚C for two weeks. The serial dilution culture under the piezophilic condition was
178
conducted twice.
179
After two weeks of incubation, the possible enrichment was examined by a
180
microscopic observation. Up to a certain dilution of culture, two morphotypes of
181
microorganisms were identified; one was a motile, thin, long spiral cell and the other
182
was a non-motile, short, oval rod. The short, oval rods were also observed in two more
183
dilution steps of culture than the one containing two morphotypes. Thus, the highest
184
dilution of culture of oval rods was further conducted to the extinction-dilution culture
185
for the purification under the same condition. For the purification of long spiral cells,
186
the highest dilution of culture including the long spiral and short oval cells were
187
inoculated into a serial dilution of MMJHS medium under a gas phase of 80% H2 +
188
20% CO2 (0.2 MPa) and incubated under the non-piezophilic condition at 50 ˚C. Under
189
the non-piezophilic culture condition, only the long spiral cells were grown. The highest
190
dilution obtained from the non-piezophilic cultivation was again applied to the serial
191
dilution cultivation under the piezophilic cultivation condition (36 MPa). This
192
procedure provided only the growth of long spiral cells under the high pressure. Thus,
193
the extinction-dilution purification of this morphotype was conducted by the repeated 9
194
cultivation under every piezophilic and non-piezophilic condition.
195
Finally, two morphotypes were isolated and were designated as strain 106 (long
196
spiral cell) and strain 108 (short oval rod), respectively. The purity was confirmed
197
routinely by microscopic examination and by repeated partial sequencing of the 16S
198
rRNA gene using several PCR primers.
199
Morphology: Cells were routinely observed under a phase-contrast Olympus
200
BX51 microscope with the Olympus DP71 digital camera system. Transmission
201
electron microscopy of negatively stained cells was carried out as described by Zillig et
202
al. (1990). Cells grown in MMJHS medium under the optimal hydrostatic pressures
203
(strains 106 and 108) or the conventional gas pressure (0.2 MPa) (strain 106) were
204
negatively stained with 2% (w/v) uranyl acetate and observed under a JEOL JEM-1210
205
electron microscope at an accelerating voltage of 80 kV.
206
Growth characteristics: Both strains were routinely cultivated at 16 MPa (for
207
strain 106) or 36 MPa (for strain 108) in Piezo-MMJHS medium, which containing 10
208
mM of dissolved H2 and 30 mM of ΣCO2 in 1 ml of modified MMJHS medium (Takai
209
et al., 2003) in a 5 ml of piezophilic cultivation syringe. The original MMJHS medium
210
contained 10 mM of sodium nitrate but the modified MMJHS medium including 20 mM
211
of sodium nitrate was used for the subsequent experiments. The pH of the medium was
212
routinely checked before, during and after the growth at a room temperature. The initial
213
pH of Piezo-MMJHS medium was adjusted to 6.5.
214
Growth was measured by direct cell counting after staining with DAPI using a
215
phase-contrast Olympus BX51 microscope. All experiments described below were
216
conducted in duplicate. To test the effects of hydrostatic pressure and temperature on
217
growth, cultures with Piezo-MMJHS medium were incubated at a varying hydrostatic 10
218
pressure of 0.2 (non-piezophilic condition), 5, 16, 36, 50 or 65 MPa at 50 ˚C and at
219
every 5 ˚C between 25 and 60 ˚C at the optimal pressures. For testing the effect of pH
220
on growth, the pH of Piezo-MMJHS medium was adjusted to lower pH than pH 6.5
221
with decreasing concentration of sodium bicarbonate and by adding HCl in MMJHS
222
medium, while was adjusted to higher pH than 6.5 with increasing concentration of
223
sodium bicarbonate and by adding NaOH in MMJHS medium. The growth was
224
conducted at 50 ˚C and the optimal pressures. The effect of NaCl on growth was test in
225
Piezo-MMJHS medium by varying a NaCl concentration in MMJHS medium.
226
The potential nutrients required for growth such as selenite, tungstate and
227
vitamins were examined with Piezo-MMJHS medium under an optimal pressure with
228
and without the specified nutrients. The nitrogen source (NH4Cl, NaNO2, N2, NaNO3
229
and yeast extract) for growth was also examined with Piezo-MMJHS medium including
230
none of the nitrogen sources under an optimum pressure. Antibiotics susceptibility was
231
tested with Piezo-MMJHS medium at an optimal pressure by using a 50 µg/ml of
232
ampicilin, kanamycin, chloramphenicol, streptomycin or vancomycin.
233
Energy and carbon sources: In an attempt to determine potential electron donors
234
for autotrophic growth of both strains, each of the H2 (10 mM), elemental sulfur (3%,
235
w/v), thiosulfate (10 mM), sulfide (1.25 mM), cystein-HCl (2.5 mM), tetrathionate (5
236
mM), and sulfite (2.5 mM) was tested instead of a combination of H2, elemental sulfur
237
and thiosulfate as a sole electron donor in Piezo-MMJHS medium (nitrate as a sole
238
electron acceptor) at 50 ˚C and an optimal pressure. In contrast, to examine potential
239
electron acceptors for autotrophic growth of both strains, each of nitrate (20 mM) and
240
O2 (0.04, 0.12, 0.4, 1.2, 2 or 4 mM) was tested instead of a combination of nitrate,
241
elemental sulfur and thiosulfate as a sole electron acceptor in MMJHS medium 11
242
(thiosulfate as a sole electron donor) at 50 ˚C and an optimal pressure. If H2 (10 mM)
243
was used as a sole electron donor, each of elemental sulfur (3% w/v), thiosulfate (10
244
mM), sulfite (2.5 mM) and ferrihydrite (5 mM) was also tested.
245
Heterotrophic growth was tested for both strains using Piezo-MMJHS medium
246
including yeast extract (0.1%, w/v), tryptone (0.1%, w/v), Casamino acid (0.1 %, w/v),
247
formate (5 mM), acetate (5 mM) or pyruvate (5 mM) instead of 30 mM of ΣCO2 at 50
248
˚C and under an optimum pressure. The pH of the media was adjusted at 6.5 in advance.
249
In addition, organotrophic growth was tested using Piezo-MMJHS medium including
250
yeast extract (0.1%, w/v), tryptone (0.1%, w/v), Casamino acid (0.1 %, w/v), formate (5
251
mM), acetate (5 mM) or pyruvate (5 mM) at pH 6.5 instead of H2, thiosulfate, elemental
252
sulfur and 30 mM of ΣCO2 at 50 ˚C and under an optimal pressure.
253
Time course of oxidations of H2 (strain 108) and thiosulfate (strains 106 and 108)
254
by nitrate-reduction and concomitant growth of both strains were examined with
255
Piezo-MMJHS medium as H2 or thiosulfate as a sole electron donor, of which all the
256
sulfate salts were replaced by the chloride salts, at 50 ˚C and under an optimal pressure.
257
The concentrations of nitrate, thiosulfate and sulfate were analyzed by ion
258
chromatography using a Shim-pack IC column (Shimadzu, Kyoto, Japan) and the
259
concentration of H2, N2O and N2 was measured by a gas chromatography Micro GC
260
CP2002 (GL Sciences, Tokyo, Japan) with a thermal conductivity detector. Nessler's
261
reagent was employed to monitor the production of ammonium ion.
262
Cellular fatty acid composition: Cellular fatty acid composition was analyzed
263
using cells autotrophically grown at 50 ˚C under the optimal pressure and the
264
non-piezophilic condition for strain 106, and using cells harvested from the piezophilic
265
autotrophic condition (at 50 ˚C and 36 MPa) and the non-piezophilic organotrophic 12
266
condition (at 50 ˚C and 0.1 MPa) for strain 108. Lyophilized cells (30 mg) were placed
267
in a Teflon-lined, screw-capped tube containing 1 ml of anhydrous methanolic HCl and
268
heated at 100 ˚C for 3 h. The resulting fatty acid methyl esters (FAMEs) were extracted
269
twice with n-hexane and concentrated under a stream of nitrogen gas. The FAMEs were
270
analyzed using a gas chromatography-mass spectrometer (Xcalibur for Trace DSQ,
271
Thermoelectron).
272
Nucleic acid analyses: DNA was prepared as described by Marmur & Doty
273
(1962). The G+C content of DNA was determined by direct analysis of
274
deoxyribonucleotides on HPLC (Tamaoka and Komagata, 1984). The 16S rRNA gene
275
was amplified by PCR using Bac 27F and 1492R primers (DeLong, 1992; Lane, 1985).
276
The nearly complete sequences of the16S rRNA genes from both strains were directly
277
sequenced by both strands using the dideoxynucleotide chain termination method with a
278
DNA sequencer Model 3100 (Perkin Elmer/Applied Biosystems Co., Foster City, CA,
279
USA). The nearly complete sequences were manually aligned to the representative
280
sequences according to the secondary structures using ARB (Ludwig et al., 2004).
281
Phylogenetic analyses were restricted to unambiguously aligned nucleotide positions.
282
Evolutionary distance matrix analysis (using the Jukes & Cantor correlation method)
283
and
284
(http://evolution.genetics.washington.edu/phylip.html).
285
performed to provide confidence estimates for phylogenetic tree topologies.
neighbor-joining
analysis
were
performed
using
PHYLIP
Bootstrap
package
analysis
was
286
The 16S rRNA gene sequences of strains 106 and 108 were deposited in the
287
DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers
288
AB468957 and AB468958, respectively.
289
Whole cell fluorescence in situ hybridization (FISH) analysis: The whole cell 13
290
FISH analysis was conducted to estimate the cellular abundance of strains 106 and 108
291
and their relatives in the in situ chimney habitat by using the 16S rRNA gene sequences.
292
The targeted microbial populations were the whole microbial cells (DAPI-stained cell
293
fraction and both bacterial- and archaeal-probes-binding cell fraction), the strain 106
294
cells (a specific probe-binding cell fraction) and the strain 108 cells (a specific
295
probe-binding fraction), respectively. The EUB338 (Stahl and Amann, 1991) and the
296
ARC915 (Amann et al., 1990) probes were used for the detection of both bacterial and
297
archaeal populations. The ribosomal RNA-targeted oligonucleotide probes specifically
298
binding to the 16S rRNAs of strains 106 and 108, respectively, were designed using
299
ARB (Ludwig et al., 2004) based on the multiply aligned sequences including those of
300
strains 106 and 108. Among several potential sites for the probes, the probes designated
301
as
302
(5’-TGGTAACTGAGGGCGTGGGT-3’) were finally chosen through the sequence
303
specificity analysis using the BLAST program to all the nucleic acid sequences in the
304
DDBJ/EMBL/GenBank nucleotide sequence databases. In the in silico analysis, the
305
TF1110 probe was found to have at least three bases of mismatch with any other 16S
306
rRNA sequences and the PB1001 probe also had more than three bases of mismatch
307
with any other 16S rRNA sequences except for the 16S rRNAs of Methanobrevibacter
308
spp. (one mismatch).
TF1110
(5’-CTCCATCTCTGGAGCCTTCC-3’)
and
PB1001
309
The whole cell FISH experiments were performed as previously described
310
(Sekiguchi et al., 1999). The frozen formalin-fixed chimney subsample was thawed, and
311
then vigorously suspended with a vortex mixer. After 5 min of static state, 0.5 ml of
312
formalin-fixed supernatant was centrifuged at a 15000 rpm at 4 ˚C for 30 min. After
313
washing with a 0.5 ml of PBS (pH 7.2) twice, the microbial cells were immobilized on a 14
314
positive charged
glass slide.
Hybridization
315
Alexa488-labeled EUB338 and the ARC915 probes and with each of the Cy3-labeled
316
specific probes TF1110 and PB1001 at 46 ˚C for 3 h. The hybridization stringency was
317
adjusted with varying concentrations of formamide in the hybridization buffer (30% for
318
the ARC915, TF1110 and PB1001 probes, and 10% for the EUB338 probe). After the
319
hybridization and the washing, the cells were stained with PBS (pH 7.2) containing
320
DAPI (10 µg/ml) for 30 min. The slide was examined under an Olympus BX51
321
epifluorescence microscopy with the Olympus DP71 digital camera system. The cells of
322
E. coli strain K12 and the other strain cells of strains 106 and 108 were used as the
323
negative controls. An average of the ratio of probe-hybridized cells to the DAPI-stained
324
cells was determined from more than 100 microscopic fields.
325
15
was performed with
both the
325
RESULTS AND DISCUSSION
326 327
Chimney habitats and total, viable and FISH cell counts: At the present, it is still
328
very difficult to determine directly the physical and chemical conditions of the
329
microbial habitats in the chimney structure by using some of the in situ hardware such
330
as temperature-, pH- and other chemical-sensors at the deep seafloor. Thus, the potential
331
chemical conditions for a given temperature range of chimney habitat have been often
332
extrapolated by the mixing between the hot endmember hydrothermal fluid and the cold
333
ambient seawater (McCollom and Shock, 1997; Shock and Holland, 2004; Tivey,
334
2004).
335
According to the chemical composition of the endmember hydrothermal fluid in
336
the BSC of the TAG field already reported (Campbell et al., 1988; Charlou et al., 1996;
337
Chiba et al., 2001), the potential concentration ranges of gaseous energy and carbon
338
substrates in the chimney habitats were estimated. If the microbially habitable
339
temperature range was assumed to be up to ~130 ˚C that could be brought by a simple
340
mixing of approx. 1:2 ratio between the hydrothermal fluid and the seawater in the case
341
of the TAG field, the available concentrations of dissolved H2, N2, CO2, CH4 and H2S
342
are estimated to be up to 0.12, 0.3, 3.4, 0.05 and 2.3 mM, respectively, based on the
343
highest values reported by Campbell et al. (1988) and Charlou et al. (1996). The total
344
dissolved concentrations of H2, ΣCO2 and reduced sulfur compounds were about 10, 30
345
and 10 mM in Piezo-MMJHS medium and 1000 –
